The role of full-length apoE in clearance of Gram-negative bacteria and their endotoxins.

ApoE is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the antibacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models. The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy, and CD spectroscopy followed by calculation of the α-helical content. The binding affinity of apoE to endotoxins was also confirmed by fluorescent spectroscopy detecting the quenching and shifting of tryptophan intrinsic fluorescence. We showed that apoE exhibits antibacterial activity particularly against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. ApoE protein folding was affected by binding of bacterial endotoxin components such as lipopolysaccharide (LPS) and lipid A, yielding similar increases in the apoE α-helical content. Moreover, high-molecular-weight complexes of apoE were formed in the presence of LPS, but not to the same extent as with lipid A. Together, our results demonstrate the ability of apoE to kill Gram-negative bacteria, interact with their endotoxins, which leads to the structural changes in apoE and the formation of aggregate-like complexes.

Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products.

Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, in silico molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria E. coli and P. aeruginosa were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with P. aeruginosa, rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria in vitro to attenuation of inflammation and bacterial levels in vivo.

Thrombin-Derived C-Terminal Peptide Reduces Candida-Induced Inflammation and Infection In Vitro and In Vivo.

Infections due to the opportunistic fungus Candida have been on the rise in the last decades, especially in immunocompromised individuals and hospital settings. Unfortunately, the treatments available today are limited. Thrombin-derived C-terminal peptide (TCP-25) is an antimicrobial peptide (AMP) with antibacterial and immunomodulatory effects. In this work, we, for the first time, demonstrate the ability of TCP-25 ability to counteract Candida in vitro and in vivo. Using a combination of viable count assay (VCA), radial diffusion assay (RDA), and fluorescence and transmission electron microscopy analyses, TCP-25 was found to exert a direct fungicidal activity. An inhibitory activity of TCP-25 on NF-κB activation induced by both zymosan alone and heat-killed C. albicans was demonstrated in vitro using THP-1 cells, and in vivo using NF-κB reporter mice. Moreover, the immunomodulatory property of TCP-25 was further substantiated in vitro by analyzing cytokine responses in human blood stimulated with zymosan, and in vivo employing a zymosan-induced peritonitis model in C57BL/6 mice. The therapeutic potential of TCP-25 was demonstrated in mice infected with luminescent C. albicans. Finally, the binding between TCP-25 and zymosan was investigated using circular dichroism spectroscopy and intrinsic fluorescence analysis. Taken together, our results show that TCP-25 has a dual function by inhibiting Candida as well as the associated zymosan-induced inflammation. The latter function is accompanied by a change in secondary structure upon binding to zymosan. TCP-25, therefore, shows promise as a novel drug candidate against Candida infections.

Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism.

Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which bind to and form amorphous amyloid-like aggregates with both bacterial lipopolysaccharide (LPS) and gram-negative bacteria. In silico molecular modeling using atomic resolution and coarse-grained simulations corroborates our experimental observations, altogether indicating increased aggregation through LPS-mediated intermolecular contacts between clusters of TCP molecules. Upon bacterial aggregation, recombinantly produced TCPs induce permeabilization of Escherichia coli and phagocytic uptake. TCPs of about 11 kDa are present in acute wound fluids as well as in fibrin sloughs from patients with infected wounds. We noted aggregation and colocalization of LPS with TCPs in such fibrin material, which indicates the presence of TCP-LPS aggregates under physiological conditions. Apart from identifying a function of proteolyzed thrombin and its fragments, our findings provide an interesting link between the coagulation system, innate immunity, LPS scavenging, and protein aggregation/amyloid formation.

Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations.

BACKGROUND: Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) is a key protein for the transport of cholesterol from the vascular wall to the liver. The formation and structure of nascent HDL, composed of apoA-I and phospholipids, is critical to this process. METHODS: The HDL was assembled in vitro from apoA-I, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a 1:4:50 molar ratio. The structure of HDL was investigated in vitreous samples, frozen at cryogenic temperatures, as well as in negatively stained samples by transmission electron microscopy. Low resolution electron density maps were next used as restraints in biased Monte Carlo simulations of apolipoprotein A-I dimers, with an initial structure derived from atomic resolution X-ray structures. RESULTS: Two final apoA-I structure models for the full-length structure of apoA-I dimer in the lipid bound conformation were generated, showing a nearly circular, flat particle with an uneven particle thickness. CONCLUSIONS: The generated structures provide evidence for the discoidal, antiparallel arrangement of apoA-I in nascent HDL, and propose two preferred conformations of the flexible N-termini. GENERAL SIGNIFICANCE: The novel full-length structures of apoA-I dimers deepens the understanding to the structure-function relationship of nascent HDL with significance for the prevention of lipoprotein-related disease. The biased simulation method used in this study provides a powerful and convenient modelling tool with applicability for structural studies and modelling of other proteins and protein complexes.

Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure.

Alzheimer's disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aß) peptides. Soluble oligomers of the Aß peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimer's disease. Single particle analyses of Aß oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aß) prevent and disrupt oligomeric assemblies of Aß in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aß shows a continuous, progressive change over a 24-hour period, while the spectrum of Aß treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aß within the oligomer provides a complementary determinant of Aß toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aß, it does induce a net reduction in beta secondary content compared to untreated samples of Aß. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aß oligomers and disrupt existing oligomers, while retaining Aß as a population of smaller, yet largely disordered oligomers.

Molecular crowding impacts the structure of apolipoprotein A-I with potential implications on in vivo metabolism and function.

The effect molecular crowding, defined as the volume exclusion exerted by one soluble inert molecule upon another soluble molecule, has on the structure and self-interaction of lipid-free apoA-I were explored. The influence of molecular crowding on lipid-free apoA-I oligomerization and internal dynamics has been analyzed using electron paramagnetic resonance (EPR) spectroscopy measurements of nitroxide spin label at selected positions throughout the protein sequence and at varying concentrations of the crowding agent Ficoll-70. The targeted positions include sites previously shown to be sensitive for detecting intermolecular interaction via spin-spin coupling. Circular dichroism was used to study secondary structural changes in lipid-free apoA-I imposed by increasing concentrations of the crowding agent. Crosslinking and SDS-PAGE gel analysis was employed to further characterize the role molecular crowding plays in inducing apoA-I oligomerization. It was concluded that the dynamic apoA-I structure and oligomeric state was altered in the presence of the crowding agent. It was also found that the C-terminal was slightly more sensitive to molecular crowding. Finally, the data described the region around residue 217 in the C-terminal domain of apoA-I as the most sensitive reporter of the crowding-induced self-association of apoA-I. The implications of this behavior to in vivo functionality are discussed. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 683-692, 2016.

ApoA-I Milano stimulates lipolysis in adipose cells independently of cAMP/PKA activation.

ApoA-I, the main protein component of HDL, is suggested to be involved in metabolic homeostasis. We examined the effects of Milano, a naturally occurring ApoA-I variant, about which little mechanistic information is available. Remarkably, high-fat-fed mice treated with Milano displayed a rapid weight loss greater than ApoA-I WT treated mice, and a significantly reduced adipose tissue mass, without an inflammatory response. Further, lipolysis in adipose cells isolated from mice treated with either WT or Milano was increased. In primary rat adipose cells, Milano stimulated cholesterol efflux and increased glycerol release, independently of ß-adrenergic stimulation and phosphorylation of hormone sensitive lipase (Ser563) and perilipin (Ser522). Stimulation with Milano had a significantly greater effect on glycerol release compared with WT but similar effect on cholesterol efflux. Pharmacological inhibition or siRNA silencing of ABCA1 did not diminish Milano-stimulated lipolysis, although binding to the cell surface was decreased, as analyzed by fluorescence microscopy. Interestingly, methyl-ß-cyclodextrin, a well-described cholesterol acceptor, dose-dependently stimulated lipolysis. Together, these results suggest that decreased fat mass and increased lipolysis following Milano treatment in vivo is partly explained by a novel mechanism at the adipose cell level comprising stimulation of lipolysis independently of the canonical cAMP/protein kinase A signaling pathway.

Single injections of apoA-I acutely improve in vivo glucose tolerance in insulin-resistant mice.

AIMS/HYPOTHESIS: Apolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys). METHODS: Male C57Bl6 mice on a high-fat diet for 2 weeks that received a single injection of human apoA-I proteins (wild-type and Milano) were analysed for blood glucose and insulin levels during a 3 h incubation followed by glucose tolerance tests. Incorporation of injected human apoA-I protein into HDLs was analysed by native gel electrophoresis. RESULTS: ApoA-I treatment significantly improved insulin secretion and blood glucose clearance in the glucose tolerance test, with an efficiency exceeding that of lean control animals, and led to decreased basal glucose during the 3 h incubation. Notably, the two apoA-I variants triggered insulin secretion and glucose clearance to the same extent. CONCLUSIONS/INTERPRETATION: ApoA-I treatment leads to insulin- and non-insulin-dependent effects on glucose homeostasis. The experimental model of short-term (2 weeks) feeding of a high-fat diet to C57Bl6 mice provides a suitable and time-efficient system to unravel the resulting tissue-specific mechanisms of acute apoA-I treatment that lead to improved glucose homeostasis.

Binding of apolipoprotein E inhibits the oligomer growth of amyloid-ß peptide in solution as determined by fluorescence cross-correlation spectroscopy.

One of the primary neuropathological hallmarks of Alzheimer disease is the presence of extracellular amyloid plaques resulting from the aggregation of amyloid-ß (Aß) peptides. The intrinsic disorder of the Aß peptide drives self-association and progressive reordering of the conformation in solution, and this dynamic distribution of Aß complicates biophysical studies. This property poses a challenge for understanding the interaction of Aß with apolipoprotein E (apoE). ApoE plays a pivotal role in the aggregation and clearance of Aß peptides in the brain, and the ε4 allele of APOE is the most significant known genetic modulator of Alzheimer risk. Understanding the interaction between apoE and Aß will provide insight into the mechanism by which different apoE isoforms determine Alzheimer disease risk. Here we applied alternating laser excitation fluorescence cross-correlation spectroscopy to observe the single molecule interaction of Aß with apoE in the hydrated state. The diffusion time of freely diffusing Aß in the absence of apoE shows significant self-aggregation, whereas in the presence of apoE, binding of the protein results in a more stable complex. These results show that apoE slows down the oligomerization of Aß in solution and provide direct insight into the process by which apoE influences the deposition and clearance of Aß peptides in the brain. Furthermore, by developing an approach to remove signals arising from very large Aß aggregates, we show that real-time single particle observations provide access to information regarding the fraction of apoE bound and the stoichiometry of apoE and Aß in the complex.

Discoidal HDL and apoA-I-derived peptides improve glucose uptake in skeletal muscle.

Lipid-free apoA-I and mature spherical HDL have been shown to induce glucose uptake in skeletal muscle. To exploit apoA-I and HDL states for diabetes therapy, further understanding of interaction between muscle and apoA-I is required. This study has examined whether nascent discoidal HDL, in which apoA-I attains a different conformation from mature HDL and lipid-free states, could induce muscle glucose uptake and whether a specific domain of apoA-I can mediate this effect. Using L6 myotubes stimulated with synthetic reconstituted discoidal HDL (rHDL), we show a glucose uptake effect comparable to insulin. Increased plasma membrane GLUT4 levels in ex vivo rHDL-stimulated myofibers from HA-GLUT4-GFP transgenic mice support this observation. rHDL increased phosphorylation of AMP kinase (AMPK) and acetyl-coA carboxylase (ACC) but not Akt. A survey of domain-specific peptides of apoA-I showed that the lipid-free C-terminal 190-243 fragment increases plasma membrane GLUT4, promotes glucose uptake, and activates AMPK signaling but not Akt. This may be explained by changes in α-helical content of 190-243 fragment versus full-length lipid-free apoA-I as assessed by circular dichroism spectroscopy. Discoidal HDL and the 190-243 peptide of apoA-I are potent agonists of glucose uptake in skeletal muscle, and the C-terminal α-helical content of apoA-I may be an important determinant of this effect.

Characterization of the biochemical and biophysical properties of the Saccharomyces cerevisiae phosphate transporter Pho89.

In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na(+)/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.

SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade.

Accumulating evidence indicates a potential role for bacterial lipopolysaccharide (LPS) in the overactivation of the immune response during SARS-CoV-2 infection. LPS is recognized by Toll-like receptor 4, mediating proinflammatory effects. We previously reported that LPS directly interacts with SARS-CoV-2 spike (S) protein and enhances proinflammatory activities. Using native gel electrophoresis and hydrogen-deuterium exchange mass spectrometry, we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the S protein. Molecular simulations validated by a microscale thermophoresis binding assay revealed that LPS binds to the S2 pocket with a lower affinity compared to S1, suggesting a role as an intermediate in LPS transfer. Congruently, nuclear factor-kappa B (NF-κB) activation in monocytic THP-1 cells is strongly boosted by S2. Using NF-κB reporter mice followed by bioimaging, a boosting effect was observed for both S1 and S2, with the former potentially facilitated by proteolysis. The Omicron S variant binds to LPS, but with reduced affinity and LPS boosting in vitro and in vivo. Taken together, the data provide a molecular mechanism by which S protein augments LPS-mediated hyperinflammation.

Selective protein aggregation confines and inhibits endotoxins in wounds: Linking host defense to amyloid formation.

Bacterial lipopolysaccharide (LPS) induces rapid protein aggregation in human wound fluid. We aimed to characterize these LPS-induced aggregates and their functional implications using a combination of mass spectrometry analyses, biochemical assays, biological imaging, cell experiments, and animal models. The wound-fluid aggregates encompass diverse protein classes, including sequences from coagulation factors, annexins, histones, antimicrobial proteins/peptides, and apolipoproteins. We identified proteins and peptides with a high aggregation propensity and verified selected components through Western blot analysis. Thioflavin T and Amytracker staining revealed amyloid-like aggregates formed after exposure to LPS in vitro in human wound fluid and in vivo in porcine wound models. Using NF-κB-reporter mice and IVIS bioimaging, we demonstrate that such wound-fluid LPS aggregates induce a significant reduction in local inflammation compared with LPS in plasma. The results show that protein/peptide aggregation is a mechanism for confining LPS and reducing inflammation, further emphasizing the connection between host defense and amyloidogenesis.

Bioactive Suture with Added Innate Defense Functionality for the Reduction of Bacterial Infection and Inflammation.

Surgical site infections (SSI) are a clinical and economic burden. Suture-associated SSI may develop when bacteria colonize the suture surface and form biofilms that are resistant to antibiotics. Thrombin-derived C-terminal peptide (TCP)-25 is a host defense peptide with a unique dual mode of action that can target both bacteria and the excessive inflammation induced by bacterial products. The peptide demonstrates therapeutic potential in preclinical in vivo wound infection models. In this study, the authors set out to explore whether TCP-25 can provide a new bioactive innate immune feature to hydrophilic polyglactin sutures (Vicryl). Using a combination of biochemical, biophysical, antibacterial, biofilm, and anti-inflammatory assays in vitro, in silico molecular modeling studies, along with experimental infection and inflammation models in mice, a proof-of-concept that TCP-25 can provide Vicryl sutures with a previously undisclosed host defense capacity, that enables targeting of bacteria, biofilms, and the accompanying inflammatory response, is shown.

Sustained delivery of a heterodimer bone morphogenetic protein-2/7 via a collagen hydroxyapatite scaffold accelerates and improves critical femoral defect healing.

Despite the glimmer of hope provided by the discovery and commercialization of bone morphogenetic protein-2 (BMP-2) as a bone graft substitute, side effects related to the use of supraphysiological doses have hindered its clinical usage. In this study, we compared the osteoinductive potential of BMP-2 homodimer with a heterodimer of BMP-2/7, both delivered via a collagen-hydroxyapatite (CHA) scaffold delivery system, with the aim to reduce the overall therapeutic BMP doses and the associated side-effects. We first show that the incorporation of hydroxyapatite in collagen-based BMP delivery systems is pivotal for achieving efficient BMP sequestration and controlled release. Using an ectopic implantation model, we then showed that the CHA+BMP-2/7 was more osteoinductive than CHA+BMP-2. Further evaluation of the molecular mechanisms responsible for this increased osteoinductivity at an early stage in the regeneration process indicated that the CHA+BMP-2/7 enhanced progenitor cell homing at the implantation site, upregulated the key transcriptomic determinants of bone formation, and increased the production of bone extracellular matrix components. Using fluorescently labelled BMP-2/7 and BMP-2, we demonstrated that the CHA scaffold provided a long-term delivery of both molecules for at least 20 days. Finally, using a rat femoral defect model, we showed that an ultra-low dose (0.5 µg) of BMP-2/7 accelerated fracture healing and performed at a level comparable to 20-times higher BMP-2 dose. Our results indicate that the sustained delivery of BMP-2/7 via a CHA scaffold could bring us a step closer in the quest for the use of physiological growth factor doses in fracture healing. STATEMENT OF SIGNIFICANCE: • Incorporation of hydroxyapatite (HA) in a collagen scaffold dramatically improves bone morphogenic protein (BMP) sequestration via biophysical interactions with BMP, thereby providing more controlled BMP release compared with pristine collagen. • We then investigate the molecular mechanisms responsible for increased osteoinductive potential of a heterodimer BMP-2/7 with is clinically used counterpart, the BMP-2 homodimer. • The superior osteoinductive properties of BMP-2/7 are a consequence of its direct positive effect on progenitor cell homing at the implantation site, which consequently leads to upregulation of cartilage and bone related genes and biochemical markers. • An ultra-low dose of BMP-2/7 delivered via a collagen-HA (CHA) scaffold leads to accelerated healing of a critical femoral defect in rats while a 20-times higher BMP-2 dose was required to achieve comparable results.

Targeting Toll-like receptor-driven systemic inflammation by engineering an innate structural fold into drugs.

There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.

The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils.

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased ß-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t(1/2)) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.

SARS-CoV-2 spike protein aggregation is triggered by bacterial lipopolysaccharide.

SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein-LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.

Antibacterial and Anti-Inflammatory Effects of Apolipoprotein E.

Apolipoprotein E (APOE) is a lipid-transport protein that functions as a key mediator of lipid transport and cholesterol metabolism. Recent studies have shown that peptides derived from human APOE display anti-inflammatory and antimicrobial effects. Here, we applied in vitro assays and fluorescent microscopy to investigate the anti-bacterial effects of full-length APOE. The interaction of APOE with endotoxins from Escherichia coli was explored using surface plasmon resonance, binding assays, transmission electron microscopy and all-atom molecular dynamics (MD) simulations. We also studied the immunomodulatory activity of APOE using in vitro cell assays and an in vivo mouse model in combination with advanced imaging techniques. We observed that APOE exhibits anti-bacterial activity against several Gram-negative bacterial strains of Pseudomonas aeruginosa and Escherichia coli. In addition, we showed that APOE exhibits a significant binding affinity for lipopolysaccharide (LPS) and lipid A as well as heparin. MD simulations identified the low-density lipoprotein receptor (LDLR) binding region in helix 4 of APOE as a primary binding site for these molecules via electrostatic interactions. Together, our data suggest that APOE may have an important role in controlling inflammation during Gram-negative bacterial infection.